PROJECT ABSTRACT / SUMMARY Influenza A viruses (IAV) are typically described as having a near-limitless capacity to evolve because their population sizes are often large, their mutation rates are high, and their generation times are short. Indeed, IAV evolution on the global scale is characterized by the acquisition and fixation of mutations that facilitate es- cape from human immunity. These global patterns are contrasted by results from whole genome sequencing studies of influenza viruses on more local scales, which find little evidence for natural selection and instead suggest that genetic drift, the stochastic fluctuation of allele frequencies, is the dominant force shaping the evolution of IAV within and between individuals. Population genetics theory predicts that genetic drift (ran- domness) acts most strongly on small populations, where natural selection is also comparatively inefficient. Transmission of IAV between hosts involves a bottleneck in which viral population size is markedly reduced, so it stands to reason that genetic drift is amplified during IAV transmission bottlenecks. I hypothesize ge- netic drift is an underappreciated barrier to the rapid evolution of seasonal IAVs because transmission bottlenecks reduce the efficiency of natural selection. To test this hypothesis, I will evaluate the cumula- tive effects of genetic drift via a series of carefully controlled bottleneck events in cell culture. I will also assess the translational relevance of these in-vitro experiments by characterizing a large dataset of natural IAV trans- mission events in human hosts. The goal of this project is to understand the impact and mechanisms of genetic drift-constrained IAV evolution in an in-vitro system and in human hosts. This proposal will take advantage of a unique molecular toolset and will employ evolutionary hypothesis test- ing to understand the role of (1) genetic drift and (2) a deleterious mutational ratchet following serial transmis- sion bottleneck events within and between individual hosts. In order to accomplish this, I propose two conceptually related but distinct aims: Aim 1 will characterize the effects of repeated bottlenecks on IAV populations under neutral (Aim 1a) and selective (Aim 1b) conditions. Aim 2 will characterize and quantify influenza transmission bottlenecks in humans. Successful completion of the proposed experiments will definitively link transmission bottlenecks to con- strained evolution of IAV viruses at the level of the individual host and will identify mechanisms underpinning the preservation and transmission of beneficial mutations in hosts, which are essential to improve current models of IAV evolution at the population level. Additionally, the proposed experiments and analyses will pro- vide me with valuable training specifically designed to guide me toward my overall career goal ? to become an independent physician-scientist.